Molecular marker sequences of cattle Cooperia species identify Cooperia spatulata as a morphotype of Cooperia punctata

Abstract

The genus Cooperia includes important parasites of ruminants and currently contains 34 accepted species. However, even for those species infecting livestock, there is a considerable lack of molecular information and many species are only identifiable using subtle morphological traits. The present study aimed to provide molecular data to allow diagnosis of Cooperia species infecting cattle. Partial sequences of two mitochondrial (cytochrome oxidase 2, 12S rRNA gene) and two nuclear genes (isotype 1 β tubulin gene including two introns, internal transcribed spacers (ITS) were obtained from morphologically identified specimens of Cooperia pectinata, Cooperia punctata and Cooperia spatulata as well as from larvae of pure Cooperia oncophora and C. punctata laboratory isolates. Pairwise identity of ITS-2 sequences was very high and it was the only region able to identify a specimen as Cooperia sp. However, the ITS-2 was unreliable for diagnosis at the species level. All other marker sequences could not unequivocally be allocated to the genus Cooperia but allowed clear species identification with the exception of the pair C. punctata/C. spatulata for which no significant differences were found for any marker sequence. Maximum-likelihood phylogenetic analyses of individual genes as well as a multi-locus analysis covering all four sequences confirmed that specimen identified as C. spatulata were randomly distributed throughout the C. punctata cluster and formed no group of their own. In contrast, the other Cooperia species formed clearly separated and statistically supported clusters. These data indicate that C. spatulata is most likely only a morphotype of C. punctata and the name should be considered a synonym. Combinations of nuclear and mitochondrial markers should be used to identify morphotypes or cryptic species to benefit from excellent barcoding properties of the latter but allowing proper phylogenetic analyses and controlling for lineage sorting that might occur for mitochondrial genotypes within a species.

Molecular analysis of polymorphic species of the genus Marshallagia (Nematoda: Ostertagiinae)

“…This indicates that ITS2 sequences are not suitable to address taxonomic or even phylogenetic questions within the genus Marshallagia. This is in agreement with other recent studies analyzing closely related strongyle nematodes showing that ITS2 is an excellent marker to identify the genus, but that closely related species differ only minimally in their ITS2 sequence and that the phylogenetic signal obtained from ITS2 sequences of Marshallagia specimen was not reliable [13,14].…”

“…Recent work on Cooperia spp. (Cooperiidae, Cooperinae) and C. minutus (Strongylidae, Cyathostominae) revealed that combined analyses of mitochondrial and nuclear marker sequences improved species identification and phylogenetic analyses [13,14].…”

A revised checklist of Cooperia nematodes (Trichostrogyloidea), common parasites of wild and domestic ruminants

“…This information is marked in Table 1. Later on, several other species were originally described, re-described, synonymised with congeners, or designated as species inquirendae (Isenstein, 1971a;Lichtenfels, 1977;Gibbons, 1981;Newton et al, 1998;Ramünke et al, 2018). For instance, Gibbons published in 1981 a merit work revising African species of the genus Cooperia.…”

“…Nevertheless, the invalidity of C. surnabada was finally accepted by Gibbons (1981), Humbert & Cabaret (1995), Lichtenfels et al (1997), Newton et al (1998), and Raműnke et al (2018) ( Table 1). Cooperia mcmasteri was initially synonymized with C. surnabada by Karamendin (1967) and Lichtenfels (1977) and then later, with C. oncophora (Ramünke et al, 2018). Two other morphologically similar species are C. spatulata and C. punctata (Table 1).…”

“…Amarante et al (2014) developed the rDNA marker specific for C. curticei. Recent study of Ramünke et al (2018) provided molecular data about two mitochondrial and two nuclear genes that allow diagnosis of four common Cooperia spp. commonly infecting cattle.…”